Nitrate and phosphate uptake kinetics of the harmful diatom Eucampia zodiacus Ehrenberg,a causative organism in the bleaching of aquacultured Porphyra thalli

The diatom Eucampia zodiacus Ehrenberg is a harmful diatom which indirectly causes bleaching of aquacultured Nori (Porphyra thalli) through competitive utilization of nutrients during bloom events. In the present study, we experimentally investigated the nitrate (N) and phosphate (P) uptake kinetics of E. zodiacus, Harima-Nada strain. Maximum uptake rates (ρ_max), which were obtained by short-term experiments, were 0.777 and 0.916 pmol cell^?1 h^?1 for nitrate and 0.244 and 0.550 pmol cell^?1 h^?1 for phosphate at 9 and 20 °C, respectively. The half-saturation constants for uptake (K_s) were 2.59 and 2.92 μM N and 1.83 and 4.85 μM P at 9 and 20 °C, respectively. Although the maximum specific uptake rate (V_max; V_max = ρ_max/Q₀, Q₀; minimum cell quota) and V_max/K_s for nitrate at 9 °C are about 1/2 of those obtained at the optimum temperature (20 °C), they are still higher than those obtained for many other phytoplankton at their optimum temperature conditions for uptake. These results suggest that E. zodiacus utilizes nitrogen efficiently at low water temperature, and it is one of the important factors causing the serious damage to Porphyra thalli by bleaching due of this species. For phosphate, the K_s values of E. zodiacus were higher than those reported for other species; the V_max and V_max/K_s values were much lower than those of other diatoms such as Skeletonema costatum (Greville) Cleve. These results suggest that E. zodiacus is disadvantaged compared to other diatom species during competitive utilization of phosphate.     

Alexandrium pacificum Litaker sp. nov (Group IV): Resting cyst distribution and toxin profile of vegetative cells in Bizerte Lagoon (Tunisia,Southern Mediterranean Sea)

A high spatial resolution sampling of Alexandrium pacificum cysts, along with sediment characteristics (% H₂O, % organic matter (OM), granulometry), vegetative cell abundance and environmental factors were investigated at 123 study stations in Bizerte Lagoon (Tunisia). Morphological examination and ribotyping of cells obtained from a culture called ABZ1 obtained from a cyst isolated in lagoon sediment confirmed that the species was A. pacificum. The toxin profile from the ABZ1 culture harvested during exponential growth phase was simple and composed of the N-sulfocarbamoyl toxins C1 (9.82 pg toxin cell⁻¹), the GTX6 (3.26 pg toxin cell⁻¹) and the carbamoyl toxin Neo-STX (0.38 pg toxin cell⁻¹). The latter represented only 2.8% of the total toxins in this strain.High abundance of A. pacificum cysts correlated with enhanced percentages of water and organic matter in the sediment. In addition, sediment fractions of less than 63 μm were examined as a favorable potential seedbed for initiation of future blooms and outbreaks of A. pacificum in the lagoon. A significant difference in the cyst distribution pattern was recorded among the lagoon's different zones, with the higher cyst abundance occurring in the inner waters. Also, no correlation due to the specific hydrodynamics of the lagoon was observed in the spatial distribution of A. pacificum cysts and vegetative cells.     

Population dynamics of the harmful diatom Eucampia zodiacus Ehrenberg causing bleachings of Porphyra thalli in aquaculture in Harima-Nada,the Seto Inland Sea,Japan

The diatom Eucampia zodiacus Ehrenberg is one of the harmful diatom species which indirectly cause bleachings of Nori (Porphyra thalli) in aquaculture through competitive utilizing of nutrients (especially nitrogen) and resultant nutrient depletion in water columns during the bloom events. The seasonal changes in environmental factors, cell density and cell size of E. zodiacus were investigated for 4 years (April 2002–December 2005) to understand the population ecology of this diatom in Harima-Nada, the eastern part of the Seto Inland Sea, Japan. Vegetative cells of E. zodiacus were usually detected year-round. Total cell densities of E. zodiacus annually peaked from mid-February to early April, and high cell densities were observed in the whole water columns during the bloom-period. Nutrient concentrations decreased with the increase of cell density of E. zodiacus, and low nutrients concentrations continued throughout the E. zodiacus bloom-period. The average cell size (length of apical axis) of E. zodiacus populations ranged from 10.8 μm to 81.2 μm, and the restoration of cell size occurred once in autumn every year just after reaching the minimum cell size. In addition, its great seasonal regularity was confirmed by the decrease and restoration of its cell size through 4-year study period. Temperature and nutrients were suitable in autumn for the growth of E. zodiacus, its blooms never occur in that season. These results strongly suggest that E. zodiacus did not have a resting stage, and it spends autumn for size restoration and starts to bloom thereafter in Harima-Nada in winter and spring, causing fishery damage to Nori aquaculture by resulting nutrient deprivation.     

Regulation of spiroimine neurotoxins and hemolytic activity in laboratory cultures of the dinoflagellate Alexandrium peruvianum (Balech & Mendiola) Balech & Tangen

Defined experimental regimes were used to determine the effects of nutrient limitation on the toxicity of Alexandrium peruvianum in batch culture. Subsamples for cell counts and spiroimine analysis at six day intervals were used to investigate the concentrations and composition of these compounds throughout growth. An erythrocyte lysis assay for hemolytic activity was performed on cell pellets and supernatants also collected every six days over the entire growth period from all treatments. From the data, growth rates, cellular spiroimine quotas and effective concentration-fifty (EC₅₀s) for cellular and supernatant associated hemolytic activity were calculated. Phosphate limitation was identified as a key regulator of toxicity in this species, yielding maximum values of 54.1 pg cell⁻¹ for 13-desmethyl spirolide C, 96.4 pg cell⁻¹ for 12-methylgymnodimine and a potent hemolytic EC₅₀ value of 7.1 × 10³ cells. The concentrations of spiroimines detected in A. peruvianum among various treatments, in addition to a unique profile of paralytic shellfish poisoning toxins, is unique in the body of microalgal literature. Because of the multiple toxin arsenal produced by this organism, the evaluation of a single toxin clearly would have underestimated the potential virulence and significance of this clone. This study provides the first evidence that growth and toxin production of A. peruvianum are influenced by altered nutrient ratios.     

Characterization of a toxic Pseudo-nitzschia spp. bloom in the Northern Gulf of Mexico associated with domoic acid accumulation in fish

A toxic bloom of Pseudo-nitzschia spp. was observed in the Alabama coastal waters of the northern Gulf of Mexico (NGOM) in June 2009 that resulted in the accumulation of domoic acid (DA) in fish. The bloom initiated following a large storm event that likely caused increased groundwater discharge 16–20 days prior to peak densities. Eleven sites, located in littoral shoreline waters and inshore embayments spanning the entire Alabama NGOM coastline, were sampled during peak densities to assess Pseudo-nitzschia species composition and toxicity, and associated water-quality parameters. Small fish (0.27–11.9 g body weight) were collected at six of these sites for analysis of DA content. High Pseudo-nitzschia spp. densities (8.27 × 10⁴–5.05 × 10⁶ cell l⁻¹) were detected at eight sites located in the littoral shoreline and particulate DA was detected at six of these littoral sites (48.0–540 pg ml⁻¹). The bloom consisted primarily (>90%) of Pseudo-nitzschia subfraudulenta, a species previously characterized as forming only a minor component of Pseudo-nitzschia assemblages and not known to produce DA. Pseudo-nitzschia spp. were at low densities or not detected at the inshore sites and DA was detected at these sites. Pseudo-nitzschia spp. density varied along an estuarine gradient, with greater densities occurring in the most saline, clear, and nutrient-poor waters. Cell density was strongly and negatively correlated with silicate (Si) concentrations and the ratios of silicate to dissolved inorganic nitrogen and phosphate (Si:DIN and Si:PO₄). Cell toxin quota was negatively correlated with phosphate, and strongly and positively correlated with the ratio of total nitrogen to total phosphorus (TN:TP). These relationships are consistent with previous observations that indicate Pseudo-nitzschia spp. density and toxicity are likely to be greater in high salinity, high irradiance, and nutrient-poor waters. DA was detected in 128 of 131 (98%) of the fish collected, which included seven primary and secondary consumer species. This is the first demonstration of trophic transfer of DA in this region of the NGOM, indicating that toxic blooms of Pseudo-nitzschia spp. in Alabama coastal waters have the potential to transfer DA to recreationally and commercially important fish species.     

Effect of environmental and nutritional factors on growth and azaspiracid production of the dinoflagellate Azadinium spinosum

Azadinium spinosum, a small dinoflagellate isolated from the North Sea, is a producer of azaspiracids (AZAs), a group of biotoxins associated with human illness following ingestion of contaminated shellfish. Using batch and continuous cultures of A. spinosum, the present study investigated the effects of different environmental and nutritional factors (salinity, temperature, photon flux density, aeration, culture media, nitrogen sources, phosphate source, and N/P ratios) on growth, maximum cell concentration, and AZA cell quota.Azadinium spinosum grew in a wide range of conditions; from 10 ̊C to 26 ̊C and salinities from 30 to 40, under irradiances ranging from 50 μmol m⁻² s⁻¹ to 250 μmol m⁻² s⁻¹, with or without aeration. Growth and maximum cell concentration were highest at a salinity of 35, at temperatures between 18 ̊C and 22 ̊C, and with aeration. Concerning AZA cell quota, the most significant effect was observed at low temperature; the AZA cell quota was more than 20 times higher at 10 ̊C (220 fg cell⁻¹) than at temperatures between 18 ̊C and 26 ̊C. A. spinosum grew on all media tested with only slight differences in growth rate and AZA cell quota. In continuous culture, lowering the concentration of nutrients (0.5 strength of a modified K-medium) in the inflow improved AZA cell quota whereas higher concentration (doubling the normal strength of K-medium) improved maximal cell concentration. A. spinosum grew on different sources of nitrogen tested (nitrate, urea, ammonium) with almost no effect on toxin cell quota and growth, except that adding ammonium caused a decrease in growth.These first experiments on Azadinium spinosum increased our knowledge on factors affecting its growth and toxin production; furthermore, these results allowed and improved particularly A. spinosum production in pilot scale photobioreactors for AZA isolation.     

Differences in the photoacclimation and photoprotection exhibited by two species of the ciguatera causing dinoflagellate genus,Gambierdiscus

In culture, Gambierdiscus spp. have been shown to prefer irradiances that are relatively low (≤250 μmol photons m⁻² s⁻¹) versus those to which they are frequently exposed to in their natural environment (>500 μmol photons m⁻² s⁻¹). Although several behavioral strategies for coping with such irradiances have been suggested, it is unclear as to how these dinoflagellates do so on a physiological level. More specifically, how do long term exposures (30 days) affect cell size and cellular chlorophyll content, and what is the photosynthetic response to short term, high irradiance exposures (up to 1464 μmol photons m⁻² s⁻¹)? The results of this study reveal that cell size and chlorophyll content exhibited by G. carolinianus increased with acclimation to increasing photon flux density. Additionally, both G. carolinianus and G. silvae exhibited reduced photosynthetic efficiency when acclimated to increased photon flux density. Photosynthetic yield exhibited by G. silvae was greater than that for G. carolinianus across all acclimation irradiances. Although such differences were evident, both G. carolinianus and G. silvae appear to have adequate biochemical mechanisms to withstand exposure to irradiances exceeding 250 μmol photons m⁻² s⁻¹ for at least short periods of time following acclimation to irradiances of up to 150 μmol photons m⁻² s⁻¹.     

Growth and domoic acid content of Pseudo-nitzschia australis isolated from northwestern Baja California,Mexico, cultured under batch conditions at different temperatures and two Si:NO3 ratios

Domoic acid (DA) poisoning in the southern part of the California Current System has been associated typically with blooms of Pseudo-nitzschia australis. The environmental variables that promote growth and DA production in the Mexican part of this system have not been identified. The present study investigated the effect of temperature and two nutrient ratios on the growth characteristics and DA content of two (BTS-1, BTS-2) P. australis strains isolated from the Pacific coast of northern Baja California peninsula, México. Of the different temperatures assayed (10, 12, 14, 15, 18 and 20 °C), the maximum cell abundance was detected at 12 °C for BTS-2 and 14 °C for BTS-1. The highest maximum specific growth rate (1.69 day⁻¹) was measured at 15 °C for BTS-2. With the exception of cells maintained at 15 °C, growth characteristics were similar in P. australis cultured in a high Si:NO₃ (2.5) or low Si:NO₃ (0.5) ratio at each temperature. Dissolved (dDA) and cellular (cDA) DA content measured at the stationary phase of growth was similar in cells cultivated at the different temperatures. No difference in cDA (between 0.11 and 1.87 pg DA cell⁻¹) was observed in cells cultivated at the two nutrient ratios. To evaluate if P. australis accumulates DA (cDA + dDA) at different stages of the culture and not only during the stationary phase of growth, the BTS-1 strain was cultivated at 14 °C and the content of this toxin was measured during culture development. The cultures were maintained at high (HL; 200 μmol quanta m⁻² s⁻¹) and low light (LL; 30 μmol quanta m⁻² s⁻¹) and in the two nutrient ratios to evaluate the effect of these variables on DA content. The photosynthetic performance and pigment concentration were measured as indicators of the physiological condition of the cells. cDA was detected in all culture conditions and during the different stages of growth. The highest DA content was measured during the lag phase of growth and it was present mainly in the medium (dDA = 70.83 pg DA cell⁻¹). Cells cultivated at HL produced more DA than LL cultured cells. P. australis cultured in HL presented lower photosynthetic rates than LL cells and had similar concentrations of photoprotective pigments and the highest maximum photosynthetic rates were detected during the lag phase of growth in all culture conditions. The results demonstrate that P. australis from northern Baja California peninsula presents a narrow temperature range for optimal growth under batch culture conditions. P. australis produce DA at different stages of growth, and DA content was related to the light intensity at which the cells were cultivated.     

Nitrogen uptake kinetics of Prymnesium parvum (Haptophyte)

The uptake rates of different nitrogen (N) forms (NO₃⁻, urea, and the amino acids glycine and glutamic acid) by N-deficient, laboratory-grown cells of the mixotrophic haptophyte, Prymnesium parvum, were measured and the preference by the cells for the different forms determined. Cellular N uptake rates (ρ_cell, fmol N cell⁻¹ h⁻¹) were measured using ¹⁵N-labeled N substrates. P. parvum showed high preference for the tested amino acids, in particular glutamic acid, over urea and NO₃⁻ under the culture nutrient conditions. However, extrapolating these rates to Baltic Seawater summer conditions, P. parvum would be expected to show higher uptake rates of NO₃⁻ and the amino acids relative to urea because of the difference in average concentrations of these substrates. A high uptake rate of glutamic acid at low substrate concentrations suggests that this substrate is likely used through extracellular enzymes. Nitrate, urea and glycine, on the other hand, showed a non-saturating uptake over the tested substrate concentration (1–40 μM-N for NO₃⁻ and urea, 0.5–10 μM-N for glycine), indicating slower membrane-transport rates for these substrates.     

Effects of phosphorus and nitrogen amendments on the growth of Egeria najas

The effect of nutrient addition on the growth of E. najas was evaluated in a dose response experiment using sand amended with phosphorus (P) and nitrogen (N), and in enrichment trials with N and P amendments to natural sediments. Plants, water and sediment came from lagoons of the Upper Paraná River Floodplain and from Itaipu Reservoir (Brazil). Relative growth rates (RGRs) of E. najas shoots, based on dry mass (DM), varied from 0.03 to 0.060 d⁻¹ for both nutrients. Root:shoot biomass ratios were related to sediment exchangeable P (r = −0.419; P = 0.03) and N (r = −0.54; P = 0.006), however root RGR was not related to sediment nutrient concentrations. When natural sediments were amended with N and P, neither shoot nor root RGRs differed among treatments for substrata from either the reservoir or the floodplain lagoons (P > 0.05). Comparison of nutrient concentrations measured in natural sediments collected from several sites in both the Upper Paraná River Floodplain (range 49–213 μg P g⁻¹ DM; 36–373 μg N g⁻¹ DM) and Itaipu Reservoir (range 43–402 μg P g⁻¹ DM; 7.9–238 μg N g⁻¹ DM) showed that sediment N and P from these systems usually exceeded minimum requirements necessary for E. najas growth, as measured in the dose response experiment. Together, these results indicate that E. najas, at least in early stages of development, responds to sediment nutrient amendments and relies upon bottom sediments to meet its N and P requirements and that for at least two Brazilian ecosystems, growth of this species is not limited by insufficient sediment N or P. Thus, reducing N and P in water is not enough to control E. najas growth in short time periods in these ecosystems.     

Influence of specific growth rate over the secretory expression of recombinant potato carboxypeptidase inhibitor in fed-batch cultures of Escherichia coli

A high cell density cultivation protocol was developed for the secretory production of potato carboxypeptidase inhibitor (PCI) in Escherichia coli. The strain BW25113 (pIMAM3) was cultured in fed-batch mode employing minimal media and an exponential feed profile where the specific growth rate was fixed by limitation of the fed carbon source (glycerol). Plasmid loss rates were found to be proportional to the specific growth rate. Distribution of PCI along the cell compartments and the culture media was also dependent on the fixed growth rate. When specific growth rate was kept at μ = 0.10 h⁻¹, 1.4 g PCI L⁻¹ were obtained when adding the product present in periplasmic extracts and supernatant fractions, with a 50% of the total expressed protein recovered from the extracellular medium. This constituted a 1.2-fold increase compared to growth at μ = 0.15 h⁻¹, and 2.0-fold compared to μ = 0.25 h⁻¹. Last, a cell permeabilization treatment with Triton X-100 and glycine was employed to direct most of the product to the culture media, achieving over 81% of extracellular PCI. Overall, our results point out that production yields of secretory proteins in fed-batch cultures of E. coli can be improved by means of process variables, with applications to the production of small disulfide-bridged proteins. Overall, our results point out that control of the specific growth rate is a successful strategy to improve the production yields of secretory expression in fed-batch cultures of E. coli, with applications to the production of small disulfide-bridged proteins.     

Recurrent vernal presence of the toxic Alexandrium tamarense/Alexandrium fundyense (Dinoflagellata) species complex in Narragansett Bay,USA

The vernal occurrence of toxic dinoflagellates in the Alexandrium tamarense/Alexandrium fundyense species complex in an enclosed embayment of Narragansett Bay (Wickford Cove, Rhode Island) was documented during 2005 and 2009–2012. This is the first report of regular appearance of the Alexandrium fundyense/Alexandrium tamarense species complex in Narragansett Bay. Thecal plate analysis of clonal isolates using SEM revealed cells morphologically consistent with both Alexandrium tamarense Lebour (Balech) and Alexandrium fundyense Balech. Additionally, molecular analyses confirmed that the partial sequences for 18S through the D1–D2 region of 28S were consistent with the identity of the two Alexandrium species. Toxin analyses revealed the presence of a suite of toxins (C1/2, B1 (GTX-5), STX, GTX-2/3. Neo, and GTX-1/4) in both Alexandrium tamarense (6.31 fmol cell⁻¹ STX equiv.) and Alexandrium fundyense (9.56 fmol cell⁻¹ STX equiv.) isolated from Wickford Cove; the toxicity of a Narragansett Bay Alexandrium peruvianum isolate (1.79 fmol cell⁻¹ STX equiv.) was also determined. Combined Alexandrium tamarense/Alexandrium fundyense abundance in Wickford Cove reached a peak abundance of 1280 cells L⁻¹ (May of 2010), with the combined abundance routinely exceeding levels leading to shellfishing closures in other systems. The toxic Alexandrium tamarense/Alexandrium fundyense species complex appears to be a regular component of the lower Narragansett Bay phytoplankton community, either newly emergent or previously overlooked by extant monitoring programs.     

Nitrate and phosphate uptake kinetics of the harmful diatom Coscinodiscus wailesii,a causative organism in the bleaching of aquacultured Porphyra thalli

The large diatom Coscinodiscus wailesii is one of the problematic species which indirectly cause bleaching damage to “Nori” (Porphyra thalli) cultivation through competitive utilization of nutrients during its bloom. In the present study, we experimentally investigated the nitrate (N) and phosphate (P) uptake kinetics of C. wailesii, Harima-Nada strain. Maximum uptake rates (ρ_max), obtained by short-term experiments, were 58.3 and 95.5 pmol cell⁻¹ h⁻¹ for nitrate and 41.9 and 59.1 pmol cell⁻¹ h⁻¹ for phosphate at 9 and 20 °C, respectively. The half saturation constants for uptake (K_s) were 2.91 and 5.08 μM N and 5.62 and 6.67 μM P at 9 and 20 °C, respectively. The ρ_max values of C. wailesii, much higher than those of other marine phytoplankton species, suggest that C. wailesii is able to take up large amounts of nutrients from the water column. On the other hand, V_max/K_s (V_max; V_max = ρ_max/Q₀, Q₀; minimum cell quota) values of C. wailesii, which is a better measure to evaluate the competitive ability for nutrient uptake, were low in dominant diatom species. This parameter indicates that C. wailesii is disadvantaged compared to other diatom species in competing for nutrients, and the decreasing nutrient concentrations from winter to spring is an important factor limiting C. wailesii blooming in early spring.     

β-carotene production from soap stock by loofa-immobilized Rhodotorula rubra in an airlift photobioreactor

In this study, the soap stock as a sole carbon source was used for growing a carotenoid producing yeast (Rhodotorula rubra). The application of soap stock resulted in increase of carotenoids yield up to 5.36 folds when compared with the grown cultures on glucose. On the best Monod equation fitted on the specific growth rate (μ) data, the maximum specific growth rate (μ_m) and half-saturation concentration (K_S) were respectively determined at 0.064 h⁻¹ and 3.26 g L⁻¹ for total fatty acids presented in soap stock. Further tests on the carotenogenesis process were carried out in a cell-immobilized airlift photobioreactor where the natural loofa sponge was used for immobilization of the cells. The performance of the bioreactor was statistically studied by the response surface methodology (RSM) where aeration rate of 0.11 vvm and light irradiation intensity of 2517 Lx provided an optimum condition for producing β-carotene with a specific production rate of 22.65 mg g_cell⁻¹ day⁻¹.     

Alteration of plankton communities and biogeochemical cycles by harmful Cochlodinium polykrikoides (Dinophyceae) blooms

Cochlodinium polykrikoides is a globally distributed, ichthyotoxic, bloom-forming dinoflagellate. Blooms of C. polykrikoides manifest themselves as large (many km²) and distinct patches with cell densities exceeding 10³ ml⁻¹ while water adjacent to these patches can have low cell densities (<100 cells ml⁻¹). While the effect of these blooms on fish and shellfish is well-known, their impacts on microbial communities and biogeochemical cycles are poorly understood. Here, we investigated plankton communities and the cycling of carbon, nitrogen, and B-vitamins within blooms of C. polykrikoides and compared them to areas in close proximity (<100 m) with low C. polykrikoides densities. Within blooms, C. polykrikoides represented more than 90% of microplankton (>20 μm) cells, and there were significantly more heterotrophic bacteria and picoeukaryotic phytoplankton but fewer Synechococcus. Terminal restriction fragment length polymorphism analysis of 16S and 18S rRNA genes revealed significant differences in community composition between bloom and non-bloom samples. Inside the bloom patches, concentrations of vitamin B₁₂ were significantly lower while concentrations of dissolved oxygen were significantly higher. Carbon fixation and nitrogen uptake rates were up to ten times higher within C. polykrikoides bloom patches. Ammonium was a more important source of nitrogen, relative to nitrate and urea, for microplankton within bloom patches compared to non-bloom communities. While uptake rates of vitamin B₁ were similar in bloom and non-bloom samples, vitamin B₁₂ was taken up at rates five-fold higher (>100 pmol⁻¹ L⁻¹ d⁻¹) in bloom samples, resulting in turn-over times of hours during blooms. This high vitamin demand likely led to the vitamin B₁₂ limitation of C. polykrikoides observed during nutrient amendment experiments conducted with bloom water. Collectively, this study revealed that C. polykrikoides blooms fundamentally change microbial communities and accelerate the cycling of carbon, some nutrients, and vitamin B₁₂.     

The effects of light intensity on the growth of Japanese Gambierdiscus spp. (Dinophyceae)

Marine toxic dinoflagellates of the genus Gambierdiscus are the causative agents of ciguatera fish poisoning (CFP), a form of seafood poisoning that is widespread in tropical, subtropical and temperate regions worldwide. The distributions of Gambierdiscus australes, Gambierdiscus scabrosus and two phylotypes of Gambierdiscus spp. type 2 and type 3 have been reported for the waters surrounding the main island of Japan. To explore the bloom dynamics and the vertical distribution of these Japanese species and phylotypes of Gambierdiscus, the effects of light intensity on their growth were tested, using a photoirradiation-culture system. The relationship between the observed growth rates and light intensity conditions for the four species/phylotypes were formulated at R > 0.92 (p < 0.01) using regression analysis and photosynthesis-light intensity (P-L) model. Based on this equation, the optimum light intensity (L_max) and the semi-optimum light intensity range (L_s-opt) that resulted in the maximum growth rate (μ_max) and ≥80% μ _max values of the four species/phylotypes, respectively, were as follows: (1) the L_max and L_s-opt of G. australes were 208 μmol photons m⁻² s⁻¹ and 91–422 μmol photons m⁻² s⁻¹, respectively; (2) those of G. scabrosus were 252 and 120–421 μmol photons m⁻² s⁻¹, respectively; (3) those of Gambierdiscus sp. type 2 were 192 and 75–430 μmol photons m⁻² s⁻¹, respectively; and (4) those of Gambierdiscus sp. type 3 were ≥427 and 73–427 μmol photons m⁻² s⁻¹, respectively. All four Gambierdiscus species/phylotypes required approximately 10 μmol photons m⁻² s⁻¹ to maintain growth. The light intensities in coastal waters at a site in Tosa Bay were measured vertically at 1 m intervals once per season. The relationships between the observed light intensity and depth were formulated using Beer’s Law. Based on these equations, the range of the attenuation coefficients at Tosa Bay site was determined to be 0.058–0.119 m⁻¹. The values 1700 μmol photons m⁻² s⁻¹, 500 μmol photons m⁻² s⁻¹, and 200 μmol photons m⁻² s⁻¹ were substituted into the equations to estimate the vertical profiles of light intensity at sunny midday, cloudy midday and rainy midday, respectively. Based on the regression equations coupled with the empirically determined attenuation coefficients for each of the four seasons, the ranges of the projected depths of L_max and L_s-opt for the four Gambierdiscus species/phylotypes under sunny midday conditions, cloudy midday conditions, and rainy midday conditions were 12–38 m and 12–54 m, 1–16 m and 1–33 m, and 0 m and 0–16 m, respectively. These results suggest that light intensity plays an important role in the bloom dynamics and vertical distribution of Gambierdiscus species/phylotypes in Japanese coastal waters.     

The relationship between Pseudo-nitzschia (Peragallo) and domoic acid in Scottish shellfish

The diatom genus Pseudo-nitzschia (Peragallo) associated with the production of domoic acid (DA), the toxin reposnsible for amnesic shellfish poisoning, is abundant in Scottish waters. A two year study examined the relationship between Pseudo-nitzschia cells in the water column and DA concentration in blue mussels (Mytilus edulis) at two sites, and king scallops (Pecten maximus) at one site. The rate of DA uptake and depuration differed greatly between the two species with M. edulis whole tissue accumulating and depurating 7 μg g⁻¹ (now expressed as mg kg⁻¹) per week. In contrast, it took 12 weeks for DA to depurate from P. maximus gonad tissue from a concentration of 68 μg g⁻¹ (now mg kg⁻¹) to <20 μg g⁻¹ (now mg kg^‐1). The DA depuration rate from P. maximus whole tissue was <5% per week during both years of the study. Correlations between the Pseudo-nitzschia cell densities and toxin concentrations were weak to moderate for M. edulis and weak for P. maximus. Seasonal diversity on a species level was observed within the Pseudo-nitzschia genus at both sites with more DA toxicity associated with summer/autumn Pseudo-nitzschia blooms when P. australis was observed in phytoplankton samples. This study reveals the marked difference in DA uptake and depuration in two shellfish species of commercial importance in Scotland. The use of these shellfish species to act as a proxy for DA in the environment still requires investigation.     

Insecticidal properties of Pimpinella anisum essential oils against the Culex quinquefasciatus and the non-target organism Daphnia magna

The efficacy of an essential oil obtained from Pimpinella anisum fruits and its major compound, trans-Anethole, was tested on the eggs, larvae and adults of Culex quinquefasciatus. While causing no significant mortality on eggs, other tested stages were very sensitive to the essential oil and trans-Anethole. LC₅₀ for the 2nd to 4th instar larvae was estimated as 26–27 μL·L^− 1 and 15–19 μL·L^− 1 for the essential oil and trans-Anethole, respectively. As for the essential oil applied on adults, LC(LD)₅₀ was estimated as 9.3 μL mL^− 1 (spray test), 1.9 μL L^− 1 (fumigation test) and 0.6 μg cm^− 2 (tarsal test), and for trans-Anethole as 8.1 μL mL^− 1 (spray test), 2.1 μL L^− 1 (fumigation test) and 0.4 μg cm^− 2 (tarsal test). The time needed to achieve 50% mortality after application of LC(LD)₉₉ of the essential oil was significantly different; for example, in larvicidal assays it ranged from 15 to 235 min depending on the larval instar, and from 9 to 180 min when applied to adults, depending on the mode of application. It was also found that temperature had an important effect on the larvicidal efficacy of the essential oil, and oviposition deterrent activity was studied.The essential oil and trans-Anethole were toxic for Daphnia magna (62–92% mortality) and significantly reduced its fertility at high concentrations (35–50 μL mL^− 1) and long exposure (48 h). However, no negative effect on Daphnia mortality or fertility was found at shorter exposure times (6 h) and/or lower concentrations (20 μL mL^− 1).Based on the results of this study, we can recommend the essential oil from P. anisum as a suitable active substance for potential botanical insecticides.     

Detection of Karenia brevis blooms on the west Florida shelf using in situ backscattering and fluorescence data

Using shipboard data collected from the central west Florida shelf (WFS) between 2000 and 2001, an optical classification algorithm was developed to differentiate toxic Karenia brevis blooms (>10⁴ cells l⁻¹) from other waters (including non-blooms and blooms of other phytoplankton species). The identification of K. brevis blooms is based on two criteria: (1) chlorophyll a concentration ≥1.5 mg m⁻³ and (2) chlorophyll-specific particulate backscattering at 550 nm ≤ 0.0045 m² mg⁻¹. The classification criteria yielded an overall accuracy of 99% in identifying both K. brevis blooms and other waters from 194 cruise stations. The algorithm was validated using an independent dataset collected from both the central and south WFS between 2005 and 2006. After excluding data from estuarine and post-hurricane turbid waters, an overall accuracy of 94% was achieved with 86% of all K. brevis bloom data points identified successfully. Satisfactory algorithm performance (88% overall accuracy) was also achieved when using underway chlorophyll fluorescence and backscattering data collected during a repeated alongshore transect between Tampa Bay and Florida Bay in 2005 and 2006. These results suggest that it may be possible to use presently available, commercial optical backscattering instrumentation on autonomous platforms (e.g. moorings, gliders, and AUVs) for rapid and timely detection and monitoring of K. brevis blooms on the WFS.